Delivery system for antimethanogenic agents

ABSTRACT

A delivery system is provided to reduce methane production in animals or to improve the weight gain of an animal. Embodiments include a delivery system comprising a volatile and/or water soluble antimethanogenic agent with cyclodextrin or a cyclodextrin-like compound.

This application is a divisional of application Ser. No. 08/836,465filed Jul. 23, 1997 which is a 371 of PCT/AU95/00733 filed Nov. 3, 1995.

The present invention relates to a delivery system for delivering avolatile and/or water soluble antimethanogenic agent to an animal, acomposition comprising the agent and methods of treatment of an animal.

BACKGROUND OF INVENTION

Microorganisms capable of generating methane are commonly found in thegut flora of animals including ruminants. The microorganisms whichproduce methane in ruminants result in the loss of energy available tothe animal and are also believed to contribute significantly togreenhouse gases.

Specifically, it has been known for many years that suppression ofmethane production in ruminants can theoretically lead to increasedproduction and much work has been undertaken to reduce methanebiosynthesis and achieve production gains in domestic animals. This hasbeen most successfully achieved by modifications to the diet of animals.Diet manipulation is only possible in a limited number of animalproduction systems and is generally only able to reduce and notcompletely suppress methane production. A number of approaches tomethane suppression in animals are being explored, not only to increaseanimal production but also to reduce the level of methane in theenvironment because of its contribution to the “greenhouse” effect.

Bromochloromethane (BMC) and some other related substances are known toshow antimethanogenic activity when administered into the rumen ofcattle and sheep but they have physical properties which makesimpractical their use as antimethanogens in livestock productionsystems, BCM for example is a volatile liquid, boiling point 69 C.,which readily evaporates. Bromoethane sulphonate, anotherantimethanogen, is difficult to administer because it is water soluble.

While the antimethanogenic properties of BCM and other antimethanogenshave been known for more than 25 years no one has identified a practicalmeans of using them to suppress methane production in livestock andobtain production increases.

Thus there has been a long felt need in the animal production field tofind a practical means of inhibiting methanogenesis in animals, inparticular ruminants.

It may also be desirable to reduce methane production in other animals.Inhibition of methane production would reduce the environmental impactof animals. In the case of domestic animals that inhabit households, itmay be desirable to reduce methane production to achieve more pleasantconditions.

Cyclodextrins are cyclic oligosaccharides which have been used in thepharmaceutical and food industries in the preparation of variousformulations incorporating active ingredients.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome at least some ofthe difficulties of the prior art in delivering antimethanogenic agentsto animals.

In work leading up to the present invention the inventors recognised thepossibility that BMC and related antimethanogenic substances might forminclusion complexes with one or more cyclodextrins although theseinclusion complexes had not been produced previously. It was alsopossible, but by no means certain, that these inclusion complexes mighthave properties which would enable them to be used as practicalantimethanogen for suppressing methane production in animals.

The inventors have now demonstrated that inclusion complexes are formedwith a number of antimethanogenic substances and show that thesecomplexes have antimethanogenic properties and physical properties whichmade them suitable for administration of livestock by several availabledelivery processes. The BCM-α-cyclodextrin inclusion complex is shownfor example to have properties particularly suitable for administrationto livestock either as a feed additive or in a controlled releasedevice, to suppress methane production and to give production benefitsin cattle and sheep.

In its broadest form the present invention provides a delivery systemfor delivering a volatile and/or water soluble antimethanogenic agent toan animal, said system comprising a volatile and/or water solubleantimethanogenic agent with a cyclodextrin or cyclodextrin-like compoundsuch that sustained release of said agent is provided.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

It has been surprisingly found by the present inventors thatcyclodextrin completed with a volatile and/or water solubleantimethanogenic agent delays release of the agent to provide prolongedor sustained release of the agent.

The term “volatile” used herein refers to the tendency of the agent toevaporate and generally refers to fluid substances.

The volatile and/or water soluble antimethanogenic agent may be anyantimethanogenic agent which is volatile and/or water soluble. The termrefers to any compound not normally in a solid state which is capable ofinhibiting methanogenesis in animals with the proviso that the compoundis physiologically acceptable to animals. Such agents includebromochloromethane and analogues thereof such as bromochloroethane orbromochloropropane and other compounds such as halothane, 2-bromoethanesulphonate and 2-bromo-2-chloroethane sulphonate.

The cyclodextrin may be selected from α-cyclodextrin, β-cyclodextrin orγ-cyclodextrin or derivatives thereof which may be naturally and/orsynthetically produced. The cyclodextrin-like compound may be anycompound capable of slowing or controlling the release of said agent andincludes mannose based ring compounds.

The term “such that sustained release of the agent is provided” meansthat the antimethanogenic agent and cyclodextrin or cyclodextrin-likecompound are capable of dissociation whereupon the agent is released ata reduced rate compared to the agent administered on its own.

Accordingly in one aspect the invention provides an antimethanogeniccomposition for use in animals comprising a volatile and/or watersoluble antimethanogenic agent together with a cyclodextrin or acyclodextrin-like compound such that sustained release of the agent isprovided.

The term “animals” used above refers to any animal in which it isdesirable to deliver an antimethanogenic compound. Preferably theanimals are mammals. The mammals will generally be domestic animals suchas ruminants (cattle, sheep, goats, deer, elk, alpacas, llamas), andother animals including horses, pigs, dogs, cats, humans.

The antimethanogenic agent is at least partly enclosed in, confined by,or encapsulated by the cyclodextrin or cyclodextrin-like compound. Oncethe composition is administered the agent dissociates from thecyclodextrin or cyclodextrin-like compound. Preferably the cyclodextrinforms an inclusion complex with the agent.

Preferably the composition is a pharmaceutical or veterinary compositionin order to comply with the various regulatory standards for suchcompositions in different countries.

The dose of the antimethanogenic agent is in the range from about 1 to150 mg per kg. More preferably the dose range is from 1 to 120 mg perkg, still more preferably 1 to 50 mg, even more preferably from 1 to 20mg, even more preferably from 1 to 10 mg per kg of body weight.

Preferably the composition is in a particulate form. More preferably theparticles are 50-100 mesh BSM.

Alternatively preferably the composition may be in the form of a capsulesuch as a gelatin capsule or the capsules described in AustralianPatents 520409, 558009 and 555998.

In another aspect the invention provides an animal feed comprising thecomposition of the invention described above together with a nutrientsource. The animal feed may be for a vegetarian animal such as cows,sheep, etc. or for carnivores or omnivores such as pigs and dogs.

In one particularly preferred aspect the composition of the presentinvention is incorporated into lucerne pasture, hay, cereals, legumes,by-products from food industries and/or polenta in the case of domesticanimals such as cattle. In the case of dogs and cats the composition maybe incorporated into dried pet or moist pet food. The animal feed or petfood may comprise other active ingredients in addition to thecomposition of the present invention. Such active ingredients includehormones, particularly growth hormones such as hormonal growthpromotant, antibacterial compounds such as ionophores including Rumensinand the like.

In another aspect the invention provides a method of producing ananti-methanogenic composition comprising a volatile and/or water solubleantimethanogenic agent together with a cyclodextrin or cyclodextrin-likecompound, said method comprising mixing said anti-methanogenic agentwith a cyclodextrin or cyclodextrin-like compound such that sustainedrelease of the agent will be provided upon administration, andoptionally bringing the composition into a suitable dosage form.

The antimethanogenic agent is at least partly enclosed in, confined by,or encapsulated by the cyclodextrin or cyclodextrin-like compound.

The composition of the present invention may be prepared by thetechnique described in Budai and Szejtli (1981).

The terms “antimethanogenic agent”, “cyclodextrin” and“cyclodextrin-like compound” have the same meaning as given above.

Those skilled in the art will be familiar with the conditions necessaryto produce the antimethanogenic composition.

In another aspect the present invention provides a method ofadministering an antimethanogen to animal over an extended periodcomprising administering a composition comprising a volatile and/orwater soluble antimethanogenic agent together with a cyclodextrin orcyclodextrin-like compound such that sustained release of the agent isprovided, in a manner such that said composition is retained by saidanimal over said period.

The term “over an extended period” refers to a period of time which islonger than the time taken for the volatile and/or water solubleantimethanogenic agent to evaporate when it is not present in thecomposition.

The term “in a manner such that said composition is retained by saidanimal over said period” means that the composition is applied in asuitable manner to allow sustained release. In a ruminant for exampleadministration may be provided in the form of a controlled releasedevice or may be provided in the feed. In monogastric animals thecomposition will be administered in the feed or alone.

Where the animal in the method is a ruminant the method leads toincreased weight gains. The method is also of benefit in both ruminantsand non-ruminants in that it reduces greenhouse gas emissions Inaddition the method is also beneficial in humans and household petswhere the antimethanogenic effects lead to reduced flatulence.

In a particularly preferred embodiment the invention relates to a methodof reducing methane production in an animal over an extended periodcomprising administering a methane reducing effective amount of anantimethanogenic composition said composition comprising volatile and/orwater soluble antimethanogenic compound together with a cyclodextrin orcyclodextrin-like compound such that sustained release of said agent isprovided.

The antimethanogenic compound is at least partly enclosed by, confinedby or encapsulated by the cyclodextrin or cyclodextrin-like compound.

In a related aspect the present invention provides a method ofprolonging methane reduction in an animal where an antimethanogeniccompound is administered to said animal, said method comprisingadministering a volatile and/or water soluble antimethanogenic compoundcomplexed with a cyclodextrin or cyclodextrin-like compound such thatsustained release is provided to said animal.

In another related aspect the present invention provides a method-ofimproving weight gain in a ruminant comprising administering to saidruminant an effective amount of the composition of the invention for atime and under conditions sufficient to allow weight gain to occur.

The inventors have found that treatment of ruminants which are being fedon heliotrope with the antimethanogenic composition of the presentinvention leads to increased rumen metabolism of heliotrine. Presumablythis leads to reduced toxicity in the animals. Thus the presentinvention also extends to a method of increasing pyrrolizidine alkaloidmetabolism in a ruminant feeding on material containing pyrrolizidinealkaloids comprising administering to said ruminant an effective amountof a volatile and/or water soluble antimethanogen which is complexedwith a cyclodextrin or cyclodextrin-like compound in accordance with theinvention.

Preferably the antimethanogenic agent is at least partly enclosed in,confined by, or encapsulated by the cyclodextrin or cyclodextrin-likecompound.

The dose of the antimethanogen may be in the range from about 1 to 150mg per kg of body weight although those skilled in the art will be ableto establish an effective administration dose for the particularapplication.

The composition may be administered by means of an intra ruminal controlrelease device, in the form of granulated powder, in animal feed, or anyother appropriate means.

In their work on the present invention, the inventors have surprisinglyfound that administration of an antimethanogen to sheep results indecreased wool fibre diameter.

Accordingly in another aspect the present invention provides a method ofreducing wool fibre diameter in a wool producing animal comprisingadministering an effective amount of an antimethanogenic agent for atime and under conditions sufficient to allow wool growth.

The term “reducing wool fibre diameter” means to reduce the diameter ofthe wool fibre compared to the diameter of the fibre produced in ananimal when not treated by the method.

The term “wool” used above refers to any natural fibre grown by ananimal and includes wool, hair and other fibres whether or not they arekeratin based.

The term “wool producing animal” includes any animal which produces woolor hair which is desirable to harvest. This includes sheep, goats(Cashmere and Angora), rabbits, alpacas and llamas and the like.Preferably the animal is a ruminant. More preferably the animal is asheep or a goat.

The term “for a time and under conditions sufficient to allow woolgrowth” means that the treatment must be carried out for a sufficientlength of time, in the wool growing season (where applicable) and underadequate nutritional and other conditions to allow the animal to producewool. Preferably the treatment is carried out over an extended period oftime so that the wool grown is of a reduced diameter along its length.

The antimethanogenic agent may be any antimethanogenic agent. Preferablythe antimethanogenic agent is administered as part of the composition ofthe present invention, however the method is not so limited.

The dose of the antimethanogenic agent may be in the range from about 1to 150 mg per kg, preferably 1 to 50 mg per kg, more preferably 1 to 20,still more preferably 1 to 10 mg per kg of animal.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to the followingnon-limiting Figures and Examples. Specifically while the invention isexemplified with reference to ruminants the invention is understood tobe clearly applicable to non-ruminant animals.

FIG. 1 is a graph showing the amount of methane as percentage ofhydrogen and methane produced as a function of the amount of BCM (ppm)in a fixed volume of rumen fluid. The middle line represents the leastsquares best fit line. The upper and lower lines are the 95% confidencelimits.

FIGS. 2(a)-2(c). Rates of (a) liveweight gain, (b) dry matter intake and(c) efficiency of feed use for liveweight gain in weaner sheep withrestricted (R; plain) or ad libitum (U; hatched) intakes of a roughagediet in Periods 3 and 4 a and fed a supplement without (−AM; □ ) orcontaining an antimethanogen (+AM; ▪ ) during Period 3, 4 and 5. Allsheep were fed a mixed roughage and concentrate diet during Periods 2, 4b and 5.

FIGS. 3(a) and (b), Rates of change in clean wool growth, measured Using (a) sands or (b) by clipping mid-side patches, in weaner sheep withrestricted (R; plain) or ad libitum (U; hatched) intakes of a roughagediet in Period 3 and part of Period 4 (4 a) and fed a supplement without(−AM; □) or containing an antimethanogen (+AM; ▪ ) during Period 3, 4and 5. All sheep were fed a mixed roughage and concentrate diet adlibitum in Periods 2, 4 b and 5.

FIG. 4. Strength of segments of the full staple in weaner sheep withrestricted (R; plain) or ad libitum intake of a mixed roughage andconcentrate diet in Period 5 and fed a supplement without (−AM; □ ) orcontaining an antimethanogen (+AM; ▪ ) during all periods.

EXAMPLE 1 Production of Antimethanogenic Composition

The manufacture of a molecular complex between an antimethanogen andcyclodextrin modifies the physical properties of the antimethanogenturning a liquid into a stable solid which substantially reduces thevolatility of the antimethanogen and forms a stable complex. The complexacts as controlled release mechanism gradually releasing the guest as itslowly dissolves.

Materials and Methods:

Composition (complex): α-cyclodextrin (200 grams) was dissolved in water(1400 ml). The resulting solution was then vigorously stirred andbromochloromethane (BCM) (20 ml) was added. The resulting suspension wascontinuously mixed for 12 hours and then filtered and air dried atambient temperature. The white solid (220 g) was then crushed and sievedto a particle size of 50 to 100 British Standard Mesh (BSM). The complexresults in a composition containing 12% BCM and 88% cyclodextrin byweight.

A similar composition was made comprising the gas halothane which isalso an antimethanogenic agent.

Another similar composition was produced using β-cyclodextrin with BCM.

Capsule formulation: the above composition was mixed with palmitic acidand SDS (both having comparable mesh size) in a tumble mixer for 12hours and then pelleted in a pelleting machine to produce pellets ofabout 2g each. Formulations were made with 0.5% and 0.1% SDS. Eightpellets were then placed in an intraruminal capsule in accordance withAustralian Patent 558009 or 555998.

EXAMPLE 2 Study on Stability of the Complex

Materials and Methods:

The complex produced as described in Example 1 was used to determine thetolerance of the antimethanogen to environmental conditions which may beencountered in the field. The parameters tested were temperature (37, 45and 60° C.) at a relative humidity of 55%, humidity (100% at 37° C.) andsimulated sunlight (UVA and UBV irradiation) at 30° C. with a relativehumidity of 55%.

The methane inhibiting activity of the antimethanogen in an in vitrofermentation system was used as an assay for the stability studies. Theamount of antimethanogen needed to inhibit methane production in ameasured amount of rumen fluid from an animal on a set diet, wasdemonstrated not to vary significantly over a period of a month, andtherefore formed the basis for use of the rumen fluid in determining theamount of antimethanogen in a given sample. The protocol used was toplace samples containing known amounts of the antimethanogen in chambershaving the environments described above, one for each stability study.The samples were exposed to these environments for a period of up to 14weeks with samples being withdrawn and used in the in vitro assay atregular intervals. In the rumen normally about 1% hydrogen and 99%methane is produced. Antimethanogenic activity refers to the amount ofinhibition of methane production as measured by hydrogen production. Afigure of 100% antimethanogenic activity means that 100% hydrogen and nomethane is being produced. This means methanogenic activity iscompletely inhibited. A figure of 80% means that 80% hydrogen is beingproduced and 20% methane is produced. Table 1 gives the results of thestudies at various temperatures with a relative humidity of 55% and theresults from the test of stability at 30° C., relative humidity 55%under UV exposure.

TABLE 1 Stability of the complex at various temperatures with 55%relative humidity. The % expressed in the table is antimethanogenicactivity (see above for definition). Temperature 30° C. + Day 37° C. 45°C. 60° C. UV  0 100% 100% 100% 100%  7 — 100% 100% 100% 14 100% 100%100% 100% 28 100% 100% 100% 100% 42 100% 100%  95% — 49 — — — 100% 63 ——  58% — 70  80%  67% — 100% 84  66% — — — 91 —  65%  54% — 98 — — — 83%

The stability studies at 37° C. with a relative humidity of 100% (notshown) demonstrated that antimethanogenic activity continued at 100% upto 14 days and then declined to 64% at 28 days, 10% at 70 days and 5% at98 days.

Results:

From the stability studies indicate that the antimethanogen has theability to tolerate elevated temperatures for quite substantial periods.The material completely retained its activity for 42 days at 37° and45°, and completely retained its activity for 28 days at 60° C.Bromochloromethane has a boiling point of 68° C., the equivalent amountof this material placed in the above environments would evaporate inless than 12 hours. The complex material also appears to be stable whenexposed to UV irradiation (simulated sunlight) completely retaining itsactivity for 49 days, although a loss of activity beyond this point maybe in part due to the temperature in the irradiation chamber being at30° C. At very high relative humidities it appears that the complex isnot as stable.

EXAMPLE 3 Duration of Activity From a Single Dose

The incorporation of an antimethanogen into a complex enables itsactivity to be prolonged. This is due to the slow dissolution of thecomplex within the rumen of an animal and thus the gradual release ofthe active compound.

Materials & Methods:

An antimethanogenic complex in accordance with that made in Example 1was used to dose a sheep and a steer. The doses were chosen to matchprior art doses of BCM administered in the liquid form. SpecificallyTrei and Olson (1969) administered 53 mg of liquid BCM to a 40 kg sheep.This is equivalent to 440 mg of the complex of the present invention.The BCM administered by Trei & Olson (1969) resulted in antimethanogenicactivity for 15 hours. Therefore we administered a single dose of 400 mgof the complex of the present invention to a 44 kg sheep.

Johnson et al (1972) administered a single dose of 5.5 g liquid BCM to a450 kg steer. This resulted in complete inhibition of methane productionfor 6 hours. The dose used is equivalent to 46 g of the complex of thecomplex of the present invention. We administered a single dose of 5.4 gof the complex of the present invention.

Animals were given a single dose, a sample of rumen fluid was withdrawnand analysed as in Example 1.

Results:

In sheep the complex was shown to have substantial antimethanogenicactivity 24 hours after administration (less than 20% normal methaneproduction).

In cattle a single dose completely inhibited methane production in thetreated animals for at least 24 hours.

EXAMPLE 4 Inhibition of Methane Production in Vitro

Materials & Methods:

complex as produced in Example 1 was used in an in vitro assay similarto that described in Example 2.

Results:

BCM complex inhibits the production of methane in rumen fluid in vitroat a level of 5 ppm-7.5 pmm of complex and 0.6-0.9 ppm of BCM. This isshown in FIG. 1.

EXAMPLE 5 Long-term Inhibition of Methane Production in Cattle

Materials and Methods:

as per Example 3 except cattle were studied for 15 weeks and theantimethanogen was only administered up to and including week 12.

Results:

We demonstrate that the composition provides substantial methanogenicactivity over an extended period. See Table 2.

TABLE 2 Average methane production as a percentage of Hydrogen + Methanein treated cattle. Antimethanogenic Week Control Composition 0 85.8 83.51 91.5 0.5 2 85.7 3.8 3 93.4 26.3 4 66.2 1.7 5 83.6 2 6 94.9 25.1 7 96.537.1 8 96.3 38.9 9 98.6 33 10  97.8 32.1 11  97.3 32.8 12  98.4 21.8 13 92.4 29.9 14  92.9 85.1 15  98.1 97.1

EXAMPLE 6 Long-term Inhibition of Methane Production in Sheep

Materials & Methods:

similar to example 5 except that the dosages were administered in anintraruminal device at 700 mg of complex per 100 kg live weight.Specifically 16 g of formulation was delivered over a period of 43 daysat an average delivery rate of 750 mg per 100 kg per day of formulation.The formulation comprised 89.5% of the complex made as in Example 1, 10%palmitic acid and 0.5% to 1.0% sodium dodecyl sulphate.

Results:

Table 3 shows that substantial methanogenic activity was provided for upto 43 days.

TABLE 3 Average methane production showing methane inhibition in sheepwith formulation containing 0.5% SDS. Day Ratio % Hyd % Meth  0  0.00 0.31 99.69   1 217.28 99.54 0.46  3 110.05 98.87 1.13 10 292.77 99.580.42 15 258.91 99.61 0.39 18 414.81 99.76 0.24 22 391.53 99.74 0.26 25 6.33 85.21 14.79  29 282.89 99.65 0.35 32 402.82 99.75 0.25 36 328.7599.70 0.30 39 402.12 99.75 0.25 43  42.33 97.12 2.88 46  0.19 15.3984.61  50  0.14 11.88 88.12  53  0.12 11.04 88.96 

EXAMPLE 7 Changes in Rumen Volatile Fatty Acid Composition

Changes in diet quality, and associated shifts in methane production,are normally accompanied by changes in the proportions of short chainvolatile fatty acids in the rumen. Reduced acetate and increasedpropionate represents a shift from low to high molecular weight fattyacids and represents a more efficient use of nutrients in animals.

Materials & methods:

VFA were measured in accordance with standard gas liquid chromatography(GLC) analysis according to Supelco standard methods.

Cattle were fed two different diets; simulated pasture diet consistingof chaffed hay mixed with the antimethanogenic composition and a 70%concentrate diet. Animals on the 70% concentrate diet were given feedmixed with antimethanogenic composition alone (anti), Rumensin alone(Rum) and antimethanogenic composition and Rumensin (Anti and Rum) (seeTables 4 and 5). All treated animals were given a dose of 1.2 gcomplex/100 kg live weight per day. Control animals were given feedwithout any additives.

Results:

TABLE 4 Cattle weight gains on simulated pasture diet. Week TreatedControl (a) Cumulative Daily Liveweight Gains (kgs) 1 0.70 0.30 2 0.690.54 3 0.79 0.68 4 0.53 0.49 5 0.65 0.55 6 0.61 0.47 7 0.57 0.47 8 0.580.47 9 0.66 0.52 10  0.62 0.53 11  0.63 0.54 12  0.68 0.55 (b) FeedConversion Ratio 1 9.68 22.60 2 10.11 12.94 3 9.05 10.41 4 13.15 14.12 510.81 12.67 6 11.42 14.62 7 12.14 14.55 8 11.87 14.45 9 10.44 13.13 10 11.18 12.83 11  11.05 12.67 12  10.25 12.44

TABLE 5 Cattle weight gains on concentrate diet. Week Control Anti RumAnti + Rum (a) Cumulative Daily Liveweight Gains (kgs) 1 0.97 −0.18 0.57−0.47 2 0.91 0.48 0.97 0.15 3 0.93 0.66 1.01 0.52 4 0.84 0.78 1.01 0.715 0.87 0.81 0.95 0.76 6 0.89 0.65 0.82 0.77 7 0.88 0.73 0.85 0.84 8 0.920.84 0.83 0.91 9 0.95 0.92 0.91 0.96 10  0.92 0.85 0.89 0.93 11  0.970.87 0.90 0.97 12  0.96 0.92 0.94 0.98 (b) Feed Conversion Ratio 1 11.96— 16.54 — 2 12.65 15.1 9.79 38.62 3 12.46 11.96 9.33 12.25 4 13.82 10.6410.21 9.80 5 13.39 10.46 11.14 9.95 6 13.05 12.77 12.81 10.09 7 13.0911.37 12.43 9.69 8 12.51 9.98 12.49 9.17 9 12.03 9.27 11.46 8.82 10 12.37 10.25 11.66 9.24 11  11.73 10.07 11.55 8.95 12  11.75 9.65 11.149.01

Table 4 shows results obtained in the simulated pasture diet. Treatedcattle consistently gained more weight and had better feed conversionratios than untreated cattle.

Table 5 shows results obtained in the 70% concentrate trials. Theresults show that the combination of Rumensin and the antimethanogeniccomposition has an additive effect on weight gain in contrast toRumensin alone or the antimethanogenic composition alone.

EXAMPLE 8 Effects in Sheep Wool

Weaner wethers (90), ranging between 25 and 35 kg in liveweight, weselected shortly after shearing in early October, 1993. They were housedin individual pens for the following 154 days. The 154 days consisted of5 periods of 21, 28, 42, 35 and 28 days respectively; these periodsaccorded to different dietary treatments and/or the times over whichmeasurements of wool growth were carried out (see details below).Liveweights were recorded weekly and feed intakes daily during the timethe sheep were held indoors

The sheep were offered a medium-high quality, mixed roughage andconcentrate diet ad libitum for 21 days (Period 1) while acclimatisingto being held indoors. At the end of the period the ten animals with thelowest or most variable intakes were removed. For the following 28 days(Period 2) the remaining 80 sheep were fed 80% of the average of theirad libitum intake during the last 5 days of Period 1. During Period 2,and subsequent periods indoors, all sheep were also fed 125 g of asupplement, consisting of a mixture of lucerne chaff and hammer-milledlupine and oats. The supplement was always given and consumed prior tofeeding the main ration.

At the end of period 2 the sheep were randomly allocated to one of 4treatment groups (20 sheep/group), following stratification according toliveweight, liveweight gain and the rate of wool growth measured usingmid-side patches during the latter half of the period (see Measurementof wool growth, etc for details). Sheep in 2 of the 4 groups wereassigned to receive antimethanogen (AM) comprising the composition madein Example 1 in their daily supplement (+AM), with the other 2 groupsremaining untreated (−AM). For the following 42 days (Period 3) sheep inone of the untreated groups were fed restricted amounts of a lowerquality, roughage diet (Restricted; R), plus the supplement alone.Levels of intake were set, using the estimated metabolisable energycontent of the diet and liveweight, so the sheep would loseapproximately 50 g/d in liveweight. Sheep in one of the +AM groups werematched to those in the −AM group using liveweight and pair-fed the samediet plus the supplement; the supplement contained 340 mg of AM. Thisenabled a comparison of the effects of treatment on the efficiency offeed use and wool growth during liveweight loss, independent frompossible effects on intake. All intakes were adjusted downward by 25 g/don day 28 of the period to maintain liveweight losses. Sheep in theremaining A−AM and +AM groups were fed the same diet ad libitum, plustheir respective supplements (Unrestricted; U). Data from these 2 groupswere used to assess the effects of AM on feed intake as well asproduction.

Period 4 was used to examine whether AM would affect the response instaple strength to acute changes in nutrition. For the first 18 days thefeeding regimes were similar to those in Period 3, with a furtherreduction of 50 g/d in the roughage intakes of both the R groups fromday 12 on. All sheep were then fed only their respective supplements forthe following 3 day (days 19, 20 and 21). From day 22 onward, sheep inboth the U and R groups were fed ad libitum on diet 1, plus theirrespective supplements. The level of AM in the supplement of the +AMsheep was increased to 370 mg/d to account for increases in liveweightof sheep in the U groups.

All sheep continued to be fed the same diet ad libitum plus supplementsduring the 28 days of period 5. This period was used to assess again theeffects of the AM treatment on intake as well as production.Supplementation with the AM chemical ceased at the end of period 5 andthe sheep were returned to the CSIRO farm. For the following 56 days,until shorn, they grazed dry, subterranean based, annual pasture andwere supplemented 3 times a week with a mixture of oats and lupins. Thesupplementary feeding rate was equivalent to 250 g/head.day 1 with theratio of oats to lupins being progressively altered from 1:2 to 2:1 overthe first 4 weeks of supplementation.

Measurements

Rates of wool growth were assessed using both dyebands and by clippingapproximately 100 cm² patches on the mid-side. Dyebands, which wereapplied to mid-side wool at the end of each Period, were removed 7 daysprior to the sheep being shorn. Greasy wool weight (fleece+locks+belly)was recorded at shearing and a further sample of mid-side wool collectedfor determination of yield, clean wool weight and average fibre diameterby the Australian Wool Testing Authority. Wool growth rates in eachperiod were then determined using greasy wool weight and the proportionsof wool grown between dyebands in dyebanded staples. Rates of woolgrowth in Periods 2 to 6 were adjusted for yield.

Estimates of rates of wool growth using clipping were carried out morefrequently than those using dyebands to try and minimise carry-overeffects of dietary treatments. Kid-side areas were delineated at the endof Period 1 using a 10×10 cm square template. Wool was clipped from thisarea using Oster® clippers with a no. 44 blade and an accuratemeasurement made of the clipped area. Wool was reclipped from the samemidside area on days 16 and 28 of Period 2 (Periods 2 a and 2 b), days14 and 42 of Period 3 (Periods 3 a and 3 b), days 19 and 35 of Period 4(Periods 4 a and 4 b) and day 28 of Period 5 (Period 5). Wool from theclipped patches was placed in a conditioning room for 24 hours beforeweighing. It was then washed twice with hot water containing detergent,rinsed thoroughly in clean water and dried in hot air before beingplaced in a desiccator for 24 hours. Samples were then reweighed and theclean weight calculated using an 18% regain. This weight and the area ofthe clipped patch were used to calculate rates of wool growth expressedas mg clean wool grown per day per cm².

Staple length and-strength of wool from each sheep were determined using5-8 dyebanded staples and Agritest (Sydney) length and strength testinginstruments. The diameter of staples used for strength testing wasrestricted to between 0.8 and 1.1 mm. The position of break (POB),expressed as the percentage weight from tip, was measured by weighingthe two halves of staples following staple strength determinations.Dyebands were also used to make visual observations on the period ofgrowth in which the POB occurred. The time of the POB was calculatedusing the POB and rates of wool growth from dyeband measurements; linearrates of wool growth during each period were assumed.

The strength of the segments of staples grown during periods 2, 3, 4 and5 were measured by clamping dyebanded staples at the edges ofconsecutive dyebands in the jaws of the Agritest staple strength tester.The tex measurements used for each segment (period) was the average ofseveral measurements of staple diameter taken between the respectivedyebands. Only 1-2 measurements were made for each staple segmentbecause of the limited availability of dyebanded staples.

Subsamples of feed and the supplement, collected regularly during dailyfeeding, were oven dried at 90° C. for 24 h for determination of drymatter and dry matter intakes. Further samples of feed and feedingredients were air dried and ground for determination of nitrogen andin vitro digestibility.

Wool-free liveweights were estimated using liveweight and measurementsof rates of wool growth made using dyebands. These estimates were thenused to calculate the daily rate of wool-free liveweight gain (positiveor negative) for each week between liveweight measurements, for each ofthe intervals over which wool growth was measured using clipped patches,for each period and for the total time that sheep remained on the samedietary regime. The latter estimates were carried out using linearregression analysis with the weekly estimates of wool-free liveweight.These estimates excluded the first week's measurement after a change indiet or amount of feed offered so as to minimise changes in liveweightdue to gut fill. Subsequently, the efficiency of feed use for liveweightgain during each of the various time intervals was calculated as woolfree liveweight gain (+ or −) per day per g of dry matter intake.

Statistical analyses

All data, excluding dry matter intake, were analysed initially usingsimple analysis of variance with a 2 by 2 factorial design. Data fromwithin each time interval where the feeding regimes were constant werealso subjected to repeated measures analysis of variance, with andwithout covariates.

The relationship between the strength of the segment of staple in whichthe POB occurred and staple strength of the full staple was estimatedfor each treatment group using linear regression analysis. Therelationships were then compared and, since they were found to be notsignificantly different, a common relationship was derived.

Results:

Liveweight, wool-free liveweight and wool-free liveweight change.

Using WFLW2 as a covariate (P<0.01 to 0.001) sheep in the U groups weresignificantly heavier (P<0.001) at all weighing times after the start ofPeriod 3 (data not shown). These differences persisted after the sheepreturned to the field. Small differences in liveweight which developedbetween the − and +AM groups during Periods 3 and 4 a were notstatistically significant. However, during the second half of period 4(period 4 b) and in period 5 +AM sheep were heavier (P<0.05) at 5 of the7 weighings. This difference was still apparent at the last weighing inthe field in May.

Identical results were obtained with wool-free liveweights although theeffect of treatment with AM was reflected sooner in a higher wool-freeliveweight gain (FIG. 2a) in the +AM sheep during Period 4 a (P<0.05;).This was the result of a lower loss in liveweight in the R/+AM group anda higher liveweight gain in the U/+AM group. Similar effects wereobserved during Period 3 but the variability, both between weeks andbetween sheep within treatment groups, was high. Consequently, thedifference of 7 g/d in wool-free liveweight gain between the R/+AM andR/−AM groups during the entire period of restricted intake, ascalculated by linear regression, was not significant. Positive effectsof the +AM treatment on wool-free liveweight gain were also observed atthe ends of Periods 4 and 5 (P<0.05) and again in Period 6, after AMtreatment had ceased. The higher, average daily gain by the +AM sheep of6 g/d during Periods 4 b and 5 was not significant.

Feed intake

Changes in the DMI are illustrated in FIG. 2b. This figure shows clearlythat the difference in wool-free liveweight gain between the U/−AM andU/+AM groups in Period 4 a was not due to a difference in DMI. Theaverage DMI of these groups over Periods 3 and 4 a were 932 and 916 g/drespectively. DMI of the +AM sheep was likewise marginally lower thanthat of the −AM sheep during Period 5 but the difference neverapproached significance.

Efficiency of feed use

The efficiency of feed use for wool-free liveweight gain, shown in FIG.2c, tended to reflect changes in wool-free liveweight and wool-freeliveweight gain. It was not influenced by any covariate measurementsfrom Period 2. There were major effect of Intake in Periods 3, 4 and 5(P<0.01 to P<0.001). R groups tended to have the higher efficiencyduring Period 4 b and in the first 3 weeks of period 5. However, by week4 of Period 5 Intake had no effect. AM treatment lead to a small butsignificantly higher efficiency in the R group at the end of Period 3and again in Period 4 a (P<0.05). Over the time that all sheep were fedthe higher quality diet ad libitum during Periods 4 b and 5, efficiencywas marginally higher again in the R/+AM group (P=0.07).

Wool growth and quality

Rate of wool growth in Period 2 was significant when used as a covariatein analyses of rates of wool growth but had no effect with measurementsinvolving the whole fleece. On the other hand WFLW2 was significant tovarying degrees when used as a covariate in both sets of analyses.Neither greasy nor clean wool production were affected by AM treatmentalthough both were higher in the U groups (P<0.0.01) (data not shown).In contrast, Intake had no significant effect on fibre diameter butthere was significantly reduced by the AM treatment (P<0.05). Theaverage fibre diameter of +AM groups was approximately 0.7 mm lower thanthat of the −AM groups. Neither Intake nor AM treatments had any effecton staple length but the staple strength of the R groups was some 12N/ktex lower than that of the U groups (P<0.001). POB in the R groupswas also game 4.6% higher (P<0.001) This difference translated into a 10day difference in the mean time of the POB (P<0.001). The average timesof the POB in the U and R groups were day 1 and day 11 of Period 4respectively. Both these times were before the sudden changes innutrition. There was no effect of the AM treatment on staple strength,POB or time of POB.

Analysis of the rates of wool growth measured using dyebands (FIG. 3),using the rate of wool growth in period 2 as a covariate, showedsignificant effects of Intake in Periods 3 (P<0.001), 4 (P<0.001) and 5(P<0.05). Rates of wool growth were higher in the U groups of sheep fromperiod 3 onward. AM treatment was without effect. When data from allperiods from period 2 onward were analysed using repeated measuresanalysis of variance, Intake and Period of growth were significant(P<0.001), with a significant. Period of growth×Intake interaction(P<0.001).

Comparing rates of wool growth using clipped patches (FIG. 4), with therate of growth in period 2 b as a covariate, also showed that the Ugroups had higher rates of wool growth in all periods after Period 3 a.(P<0.001). Differences between the U and R groups in Period 5 were morepronounced than seen using dyeband measurements. The rate of wool growthwas also marginally lower in the +AM groups in several periods but thisdifference was significant only in Period 3 b (P<0.05). Repeatedmeasures analysis of variance again showed a significant effect ofIntake (P<0.001) and different changes in rate of growth of the U and Rgroups over time (P<0.001). A possible Period of growth×AM interactionwas also indicated (P=0.07). This appeared to arise from a relativelysharper decline in rate of growth in Period 3 a and smaller increase inPeriod 5 in the +AM group compared with the −AM.

Changes in staple strength were observed. Segment strength wasconsistently lower for the R groups in Periods 3 (P<0.01), 4 (P<0.001)and 5 (P<0.05). AM treatment had no effect. Likewise, there were noapparent effects of the changes in nutrition in Period 4. The segmentsof the staple with the lowest strength were all grown in period 5; thiscontrasted with the times of the POB. A highly significant linearrelationship was found between the strength of the full staple and thestrength of the segment in which the POB occurred (P<0.001).

The absence of any significant effect on the +AM treatment on rates ofwool growth was unexpected. The reduced fibre diameter was extremelysurprising given the absence of increased wool growth rates and the factthat there were no alterations in staple length. It was also surprisingthat sound wool was produced in the R groups despite a 7 to 8% weightloss.

EXAMPLE 9 Heliotrope Detoxification

The inhibition of methane within the rumen of sheep and cattle increasesthe availability of hydrogen to hydrogen utilising bacteria such asPeptostreptococcus heliotrinereducans, a microbe which leads to thereductive breakdown of pyrrolizidine alkaloids.

Materials & Methods:

The concentrations of heliotrine and other major pyrrolizidine alkaloidin heliotrope in the rumen of sheep were measured. The sheep were fed adiet comprising 50% heliotrope and 50% oaten-lucerne chaff given onceper day. Treated sheep were administered with a sheep capsule carryingthe previously described formulation delivering an average of 330 mg ofcomplex per sheep per day. Samples of the rumen contents were taken 24hours after feeding, frozen and then analysed for heliotrine.

Results:

A significant reduction was observed over the first 40 days in rumenpyrrolizidine alkaloid concentrations of sheep treated with theantimethanogen formulation. This is a significant effect. During thisinitial 40 day period normal untreated sheep are at their mostinefficient in terms of their ability to degrade pyrrolizidinealkaloids. In the treated sheep the antimethanogen augments thedetoxification action of P. heliotrinreducans, as described by Lanigan(1971, 1972), by making hydrogen, an apparently essential substrate inthe detoxification reaction, more available in the rumen. Compared tocontrols, treated sheep more efficiently break down the pyrrolizidinealkaloids daring the critical period when animals are first exposed tothe toxins in their diet. After a 40 day induction period, in which thedetoxification activity of P. heliotrinreducans increases, the untreatedcontrol sheep become as efficient as the treated sheep in breaking dawnpyrrolizidine alkialoids in their rumen.

EXAMPLE 10 Trials with Antimethanogen and a Hormone

Materials & Methods

The composition described in Example 1 was used as a feed additive in acattle productivity trial feeding a baled pasture diet formulated tosimulate a typical tropical wet season diet. The trial consisted of fourgroups of cattle two receiving the antimethanogenic additive and twobeing implanted with a hormone growth promotant, (Compudose 100), in atypical 2×2 trial design.

Weekly liveweight changes and daily feed intake were measured andoverall feed conversion efficiency for the treatment period wascalculated.

Results:

Live weight gains

Control animals

0.585 Kg/Day

Antimethanogen

0.617

Compudose 100

0.714

Compudose+antimethanogen

0.798

Dry Matter intake

Control animals

18.71 g/KgLW/Day

Antimethanogen

16.87

Compudose 100

19.07

Compudose+antimethanogen

18.17

Feed Conversion efficiency

Control animals

8.0 kgDM/kg liveweight

Antimethanogen

7.1

Compudose 100

6.78

Compudose+antimethanogen

6.0

Thus the animals fed the antimethanogen compudose bad better live weightgains and feed conversion efficiencies than the animals fedantimethanogen alone and the control animals.

This demonstrates that the composition of the present invention may beused in conjunction with commercially available products and stillproduce the desired effects.

References

Budai Z. S. & Szejtli J. “Recovery of solvent vapours from gaseous phaseby solvent solutions” I. Int. Symp. on Cyclodextrins, Budapest, 1981.

Lanigan, G. W. (1971) Aust. J. Agric. Res 22:123-130.

Lanigan, G. W. (1972) Aust. J. Agric. Res 23:1085-1091

Trei, J. E. & Olson, W. A. (1969) J. Anim. Sci 29:173

Johnson, E. D. et al (1972) Con. J. Anim. Sci. 52:703-712

What is claimed is:
 1. A method of reducing wool fibre diameter in awool producing animal comprising administering an effective amount of anantimethanogenic agent for a time and under conditions sufficient toallow wool growth.
 2. The method of claim 1 wherein saidantimethanogenic agent administered is the composition of claim
 1. 3.The method of claim 1 wherein the dose of the antimethanogenic agent is1 to 150 mg per kg of body weight.
 4. The method of claim 1 wherein saidanimal is selected from the group consisting of sheep and goats.
 5. Amethod of claim 1, wherein said antimethanogenic agent administered is acomposition comprising a volatile and/or water soluble antimethanogenicagent together with cyclodextrin or cyclodextrin-like compound such thatsustained release of said agent is provided.
 6. A method of producing anantimethanogenic composition, said method comprising mixing a volatileand/or water soluble antimethanogenic agent with a cyclodextrin orcyclodextrin-like compound such that sustained release of said agentwill be provided on administration and optionally bring the compositioninto a suitable dosage form.
 7. A method of administering anantimethanogenic agent to an animal over an extended period comprisingadministering a composition comprising a volatile and/or water solubleantimethanogenic agent together with a cyclodextrin or cyclodextrin-likecompound such that sustained release of said agent is provided in amanner such that said composition is retained by said animal over saidperiod.
 8. A method of claim 7, wherein said antimethanogenic agent asleast partly enclosed in, confined by or encapsulated by saidcyclodextrin or said cyclodextrin-like compound.
 9. A method of claim 7,wherein said animal is a mammal.
 10. A method of claim 7, wherein saidanimal is a domestic animal.
 11. A method of claim 9, wherein saidanimal is selected from the group consisting of a cow, a sheep, a goat,a deer, an elk, an alpaca, a llama, a horse, a cat, a dog, a pig and ahuman.
 12. A method of claim 8, wherein said antimethanogenic compoundis administered in a range from 1 to 120 mg per kg of body weight.
 13. Amethod of claim 12, wherein said compound is administered in a rangefrom 1 to 10 mg per kg of body weight.
 14. A method according to claim7, wherein the cyclodextrin or cyclodextrin-like compound isα-cyclodextrin, β-cyclodextrin or γ-cyclodextrin.
 15. A method accordingto claim 7, wherein the cyclodextrin or cyclodextrin-like compound is amannose-based ring compound.
 16. A method of reducing methane productionin an animal over an extended period comprising administering a methanereducing effective amount of an antimethanogenic composition, saidcomposition comprising a volatile and/or water soluble antimethanogeniccompound together with cyclodextrin or cyclodextrin-like compound suchthat sustained release of said agent is provided.
 17. A method ofprolonging methane reduction in an animal where a volatile and/or watersoluble antimethanogenic compound is administered to said animal, saidmethod comprising administering said antimethanogenic compound complexedwith a cyclodextrin or a cyclodextrin-like compound to said animal.